Role of Tachykinin 1 and 4 Gene-Derived Neuropeptides and the Neurokinin 1 Receptor in Adjuvant-Induced Chronic Arthritis of the Mouse

Objective

Substance P, encoded by the Tac1 gene, is involved in neurogenic inflammation and hyperalgesia via neurokinin 1 (NK1) receptor activation. Its non-neuronal counterpart, hemokinin-1, which is derived from the Tac4 gene, is also a potent NK1 agonist. Although hemokinin-1 has been described as a tachykinin of distinct origin and function compared to SP, its role in inflammatory and pain processes has not yet been elucidated in such detail. In this study, we analysed the involvement of tachykinins derived from the Tac1 and Tac4 genes, as well as the NK1 receptor in chronic arthritis of the mouse.

Methods

Complete Freund’s Adjuvant was injected intraplantarly and into the tail of Tac1^−/−, Tac4^−/−, Tacr1^−/− (NK1 receptor deficient) and Tac1^−/−/Tac4^−/− mice. Paw volume was measured by plethysmometry and mechanosensitivity using dynamic plantar aesthesiometry over a time period of 21 days. Semiquantitative histopathological scoring and ELISA measurement of IL-1β concentrations of the tibiotarsal joints were performed.

Results

Mechanical hyperalgesia was significantly reduced from day 11 in Tac4^−/− and Tacr1^−/− animals, while paw swelling was not altered in any strain. Inflammatory histopathological alterations (synovial swelling, leukocyte infiltration, cartilage destruction, bone damage) and IL-1β concentration in the joint homogenates were significantly smaller in Tac4^−/− and Tac1^−/−/Tac4^−/− mice.

Conclusions

Hemokinin-1, but not substance P increases inflammation and hyperalgesia in the late phase of adjuvant-induced arthritis. While NK1 receptors mediate its antihyperalgesic actions, the involvement of another receptor in histopathological changes and IL-1β production is suggested.     

MeltMan: Optimization,Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.     

Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium: Role of Rac and PI3K

Brain metastases are common and devastating complications of both breast cancer and melanoma. Although mammary carcinoma brain metastases are more frequent than those originating from melanoma, this latter has the highest tropism to the brain. Using static and dynamic in vitro approaches, here we show that melanoma cells have increased adhesion to the brain endothelium in comparison to breast cancer cells. Moreover, melanoma cells can transmigrate more rapidly and in a higher number through brain endothelial monolayers than breast cancer cells. In addition, melanoma cells have increased ability to impair tight junctions of cerebral endothelial cells. We also show that inhibition of Rac or PI3K impedes adhesion of breast cancer cells and melanoma cells to the brain endothelium. In addition, inhibition of Rac or PI3K inhibits the late phase of transmigration of breast cancer cells and the early phase of transmigration of melanoma cells. On the other hand, the Rac inhibitor EHT1864 impairs the junctional integrity of the brain endothelium, while the PI3K inhibitor LY294002 has no damaging effect on interendothelial junctions. We suggest that targeting the PI3K/Akt pathway may represent a novel opportunity in preventing the formation of brain metastases of melanoma and breast cancer.     

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO₂ uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.Energy security and climate change as global problems urge increased efforts to use plants as renewable energy sources both for power generation and transportation fuel production. Selected wood species, such as willows (Salix spp.), can be cultivated as short-rotation coppice for the rapid accumulation of biomass and reduction of CO₂ emission. Coppicing reinvigorates shoot growth, resulting in a special woody plant life cycle that differs from natural tree development, which takes decades. In this cultivation system, small stem cuttings are planted at high densities (15,000–25,000 ha⁻¹). In the soil, these dormant wood cuttings first produce roots and shoots that emerge from reactivated buds. During the first year, the growing shoots mature to woody stems. In the winter, these stems are cut back, and in the following spring, the cut stumps develop multiple shoots. The short-rotation coppice plantations are characterized by a very short, 2- to 3-year rotation, and the most productive varieties can produce up to 15 tons of oven-dried wood per hectare per year (Cunniff and Cerasuolo, 2011). The high-density willow plantations can also be efficiently used for heavy metal or organic phytoremediation, as reviewed by Marmiroli et al. (2011).The biomass productivity of shrub willows is largely dependent on coppicing capability, early vigorous growth, shoot growth rate and final stem height, root system size, photosynthetic efficiency, formation and composition of woody stems, water and nutrient use, as well as abiotic and biotic stress tolerance. Genetic improvement of all these traits can be based on broad natural genetic resources represented by more than 400 species in the genus Salix. More than 200 species have hybrid origins, and ploidy levels vary from diploid up to dodecaploid (Suda and Argus, 1968; Newsholme, 1992). In addition to molecular marker-assisted clone selection, intraspecific and interspecific crosses have been shown to further extend genetic variability in breeding programs for biomass yield (Karp et al., 2011).During natural diversification and artificial crossings of Salix spp., the willow genomes frequently undergo polyploidization, resulting in triploid or tetraploid allopolyploids. In triploid hybrids, both heterosis and ploidy can contribute to the improved biomass yield (Serapiglia et al., 2014). While the alloploid triploids have attracted considerable attention in willow improvement, the potentials of autotetraploid willow genotypes have not been exploited so far. As shown for other short-rotation wood species (poplar [Populus spp.], black locust [Robinia pseudoacacia], Paulownia spp., and birch [Betula spp.]), doubling the chromosome set by colchicine treatment can cause significant changes in organ morphology or growth parameters (Tang et al., 2010; Cai and Kang, 2011; Harbard et al., 2012; Mu et al., 2012; Wang et al., 2013a, 2013b). In several polyploidization protocols, the in vitro cultured tissues are exposed to different doses of colchicine or other inhibitors of mitotic microtubule function, and plantlets are differentiated from polyploid somatic cells (Tang et al., 2010; Cai and Kang, 2011). Alternatively, seeds or apical meristems of germinating seedlings can be treated with a colchicine solution (Harbard et al., 2012). Allotetraploids of poplar were produced by zygotic chromosome doubling that was induced by colchicine and high-temperature treatment (Wang et al., 2013a).Since tetraploid willow plants with 2n = 4x = 76 chromosomes are expected to represent novel genetic variability, especially for organ development and physiological parameters, a polyploidization project was initiated that was based on a highly productive diploid energy willow (S. viminalis var. Energo). Colchicine treatment of reactivated axillary buds of the in vitro-grown energy willow plantlets resulted in autotetraploid shoots and, subsequently, plants. For comparison of diploid and tetraploid variants of willow plants, digital imaging of green organs and roots was used for phenotyping. Among the tetraploid lines, genotypes were identified with improved biomass production, better photosynthetic parameters, and altered organ structure and hormone composition. The new tetraploid willow variants produced can serve as a unique experimental material to uncover key factors in biomass production in this short-rotation energy plant. In the future, these plants can also serve as crossing partners of diploid lines for the production of novel triploid energy willow genotypes.     

Comparing two methods for estimating floral resource availability for insect pollinators in semi-natural habitats

Pollinator and flowering plant interactions play a critical role in maintaining most terrestrial ecosystems, including agroecosystems. Although estimates of floral resource availability are essential to understand plant–pollinator relationships, no generally accepted methodology exists to date. We compared two methods for sampling floral resources in a single meadow. About every three days, we recorded species lists of insect-pollinated plants with abundance categories assigned to each species (hereafter referred to as scanning) and we counted the flowering shoots in 36 2 × 2 m quadrats (hereafter quadrat sampling). These methods were compared with respect to (i) the number of species detected, (ii) estimated floral resource abundance, and (iii) temporal changes in flowering. With scanning, we found more potential nectar-plant species and species were found earlier than with quadrat sampling. With the latter, abundant species were found with higher probability than the scarce. Flower abundances were correlated between the two methods. We predicted that a cover of 6.3 ± 3.6% should be used for an appropriate estimate of flower abundance in our study site, although the optimal cover probably varies across different habitats. Furthermore, flower abundance changed 6% per day compared to the flowering peak. Overall, scanning seems to be more appropriate for detecting presence and the timing of species, while quadrats may provide higher resolution for abundance estimates. Increased sampling coverage and frequency may enhance research accuracy and using scanning and quadrat sampling simultaneously may help to optimize research effort. We encourage further development of sampling protocols.     

Diversity of extremophilic bacteria in the sediment of high-altitude lakes located in the mountain desert of Ojos del Salado volcano,Dry-Andes

Ojos del Salado, the highest volcano on Earth is surrounded by a special mountain desert with extreme aridity, great daily temperature range, intense solar radiation, and permafrost from 5000 meters above sea level. Several saline lakes and permafrost derived high-altitude lakes can be found in this area, often surrounded by fumaroles and hot springs. The aim of this study was to gain information about the bacterial communities inhabiting the sediment of high-altitude lakes of the Ojos del Salado region located between 3770 and 6500 m. Altogether 11 sediment samples from 4 different altitudes were examined with 16S rRNA gene based denaturing gradient gel electrophoresis and clone libraries. Members of 17 phyla or candidate divisions were detected with the dominance of Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes. The bacterial community composition was determined mainly by the altitude of the sampling sites; nevertheless, the extreme aridity and the active volcanism had a strong influence on it. Most of the sequences showed the highest relation to bacterial species or uncultured clones from similar extreme environments.     

Proteomics Analysis of Thermoplasma Quinone Droplets

A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin‐N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D‐SDS‐PAGE/LC–MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods—column chromatography, immunoprecipitation, and LD ultracentrifugation—are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies.     

Brain size predicts behavioural plasticity in guppies (Poecilia reticulata): An experiment

Understanding how animal personality (consistent between‐individual behavioural differences) arises has become a central topic in behavioural sciences. This endeavour is complicated by the fact that not only the mean behaviour of individuals (behavioural type) but also the strength of their reaction to environmental change (behavioural plasticity) varies consistently. Personality and cognitive abilities are linked, and we suggest that behavioural plasticity could also be explained by differences in brain size (a proxy for cognitive abilities), since accurate decisions are likely essential to make behavioural plasticity beneficial. We test this idea in guppies (Poecilia reticulata), artificially selected for large and small brain size, which show clear cognitive differences between selection lines. To test whether those lines differed in behavioural plasticity, we reared them in groups in structurally enriched environments and then placed adults individually into empty tanks, where we presented them daily with visual predator cues and monitored their behaviour for 20 days with video‐aided motion tracking. We found that individuals differed consistently in activity and risk‐taking, as well as in behavioural plasticity. In activity, only the large‐brained lines demonstrated habituation (increased activity) to the new environment, whereas in risk‐taking, we found sensitization (decreased risk‐taking) in both brain size lines. We conclude that brain size, potentially via increasing cognitive abilities, may increase behavioural plasticity, which in turn can improve habituation to novel environments. However, the effects seem to be behaviour‐specific. Our results suggest that brain size likely explains some of the variation in behavioural plasticity found at the intraspecific level.